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Determination of chemical exposure over time in humans as a basis for risk assessment – focus on alkylating compounds

Assessing exposure to electrophilic compounds and metabolites is challenging due to their inherent reactivity. However, structural modifications on blood proteins and nucleic acids, forming adducts, can serve as valuable biomarkers of exposure to both exogenous and endogenous electrophiles. Hemoglobin adducts, in particular, enable evaluation of exposure to specific alkylating agents in epidemiological studies and in estimating occupational exposure, as well as in tracing reactive drug metabolites. We have applied this approach to hemoglobin adducts of low-molecular-weight alkylating compounds, including acrylamide and its metabolite glycidamide [1].

Advantages of hemoglobin (Hb) adducts as biomarkers

• Electrophiles are reactive and unstable, hence direct measurement is not optimal.
• Hb adducts of electrophiles can be measured after a relatively long period.
• Hb half-life is ~63 days in humans.
• Different electrophiles bind to N-terminal valine (Val) of Hb – allowing their characterization and measurement as Hb adducts.

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We employ high-resolution mass spectrometry (HRMS)-based methods to analyze adducts to hemoglobin. This includes applying the FIRE method [2,3], which is a development of the modified Edman degradation – the adduct is derivatized and released with the N-terminal valine tag. The corresponding analyte specific to the electrophile is enriched by SPE and quantified by LC-HRMS. HRMS data combined with expert knowledge is used for profiling and characterization of the adducts. Depending on the research question, analyses can be conducted across various species and tissue types.

[1] Quantitative analysis of exposure to electrophiles using blood samples: Method evaluation using acrylamide and glycidamide as model compound. Toxicol Lett. Vol 411, 2025.
[2] A new modified Edman procedure for analysis of N-terminal valine adducts in hemoglobin by LC-MS/MS. J Chromatogr B. Vol 878, 2010.
[3] Estimation of intake and quantification of hemoglobin adducts of acrylamide in adolescents in Sweden. Front Nutr. Vol 11, 2024.